Down-regulation of Sp1 suppresses cell proliferation, clonogenicity and the expressions of stem cell markers in nasopharyngeal carcinoma

Journal of Translational Medicine

Mu-Sheng Zeng¹  Man-Zhi Li¹  Shan Xing¹  Gui-Hong Liu²  Yan-Xian Li¹  Hong-Bo Wang¹  Hua Zhang¹  Jing-Ping Zhang¹ 

[1] Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, China;Department of Oncology, the Second Affiliated Hospital of Guangzhou medical college, Guangzhou, China

关键词: Stem cell;    Clonogenicity;    Cell proliferation;    Cell cycle;    Sp1;    Nasopharyngeal carcinoma;   

Others  :  1148581
DOI  :  10.1186/s12967-014-0222-1

 received in 2014-03-16, accepted in 2014-07-31,  发布年份 2014

【 摘 要 】

Background

Transcription factor Sp1 is multifaceted, with the ability to function as an oncogene or a tumor suppressor, depending on the cellular context. We previously reported that Sp1 is required for the transcriptional activation of the key oncogenes in nasopharyngeal carcinoma (NPC), including B-lymphoma mouse Moloney leukemia virus insertion region 1 (Bmi1) and centromere protein H (CENPH), but the role of Sp1 and its underlying mechanisms in NPC remained largely unexplored. The objective of this study was to investigate the cellular function of Sp1 and to verify the clinical significance of Sp1 as a potential therapeutic target in NPC.

Methods

The levels of Sp1 in the normal primary nasopharyngeal epithelial cells (NPECs) and NPC cell lines were analyzed by Quantitative Real-time RT-PCR (qRT-PCR) and Western blot. The location and expression of Sp1 in the NPC tissues were detected by immunohistochemistry staining (IHC). The effect of Sp1 knockdown on the cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype in NPC cells were evaluated by MTT, flow cytometry, clonogenicity analysis and sphere formation assay.

Results

The mRNA and protein levels of Sp1 were elevated in NPC cell lines than in the normal primary NPECs. Higher expression of Sp1 was found in NPC tissues with advanced clinical stage (P = 0.00036). Either inhibition of Sp1 activity by mithramycin A, the FDA-approved chemotherapeutic anticancer drug or Sp1 silencing by two distinct siRNA against Sp1 suppressed the growth of NPC cells. Mechanism analysis revealed that Sp1 silencing may suppress cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype through inducing the expression of p27 and p21, and impairing the expressions of the critical stem cell transcription factors (SCTFs), including Bmi1, c-Myc and KLF4 in NPC cells.

Conclusions

Sp1 was enriched in advanced NPC tissues and silencing of Sp1 significantly inhibited cell proliferation, clonogenicity, anchorage-independent growth and the stem-cell like phenotype of NPC cells, suggesting Sp1 may serve as an appealing drug target for NPC.

【 授权许可】

   
2014 Zhang et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Tao Q, Chan AT: Nasopharyngeal carcinoma: molecular pathogenesis and therapeutic developments. Expert Rev Mol Med 2007, 9:1-24.
• [2]Lo KW, To KF, Huang DP: Focus on nasopharyngeal carcinoma. Cancer Cell 2004, 5:423-428.
• [3]Wei WI, Sham JS: Nasopharyngeal carcinoma. Lancet 2005, 365:2041-2054.
• [4]Feng BJ, Huang W, Shugart YY, Lee MK, Zhang F, Xia JC, Wang HY, Huang TB, Jian SW, Huang P, Feng QS, Huang LX, Yu XJ, Li D, Chen LZ, Jia WH, Fang Y, Huang HM, Zhu JL, Liu XM, Zhao Y, Liu WQ, Deng MQ, Hu WH, Wu SX, Mo HY, Hong MF, King MC, Chen Z, Zeng YX: Genome-wide scan for familial nasopharyngeal carcinoma reveals evidence of linkage to chromosome 4. Nat Genet 2002, 31:395-399.
• [5]Lo YM, Chan LY, Chan AT, Leung SF, Lo KW, Zhang J, Lee JC, Hjelm NM, Johnson PJ, Huang DP: Quantitative and temporal correlation between circulating cell-free epstein-barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999, 59:5452-5455.
• [6]Chien YC, Chen JY, Liu MY, Yang HI, Hsu MM, Chen CJ, Yang CS: Serologic markers of epstein-barr virus infection and nasopharyngeal carcinoma in Taiwanese men. N Engl J Med 2001, 345:1877-1882.
• [7]Lin JC, Wang WY, Chen KY, Wei YH, Liang WM, Jan JS, Jiang RS: Quantification of plasma epstein-barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 2004, 350:2461-2470.
• [8]Xu FH, Xiong D, Xu YF, Cao SM, Xue WQ, Qin HD, Liu WS, Cao JY, Zhang Y, Feng QS, Chen LZ, Li MZ, Liu ZW, Liu Q, Hong MH, Shugart YY, Zeng YX, Zeng MS, Jia WH: An epidemiological and molecular study of the relationship between smoking, risk of nasopharyngeal carcinoma, and Epstein-Barr virus activation. J Natl Cancer Inst 2012, 104:1396-1410.
• [9]Gallicchio L, Matanoski G, Tao XG, Chen L, Lam TK, Boyd K, Robinson KA, Balick L, Mickelson S, Caulfield LE, Herman JG, Guallar E, Alberg AJ: Adulthood consumption of preserved and nonpreserved vegetables and the risk of nasopharyngeal carcinoma: a systematic review. Int J Cancer 2006, 119:1125-1135.
• [10]Chan AT, Ma BB, Lo YM, Leung SF, Kwan WH, Hui EP, Mok TS, Kam M, Chan LS, Chiu SK, Yu KH, Cheung KY, Lai K, Lai M, Mo F, Yeo W, King A, Johnson PJ, Teo PM, Zee B: Phase II study of neoadjuvant carboplatin and paclitaxel followed by radiotherapy and concurrent cisplatin in patients with locoregionally advanced nasopharyngeal carcinoma: therapeutic monitoring with plasma Epstein-Barr virus DNA. J Clin Oncol 2004, 22:3053-3060.
• [11]Niu X, Hu C, Kong L: Experience with combination of cetuximab plus intensity-modulated radiotherapy with or without chemotherapy for locoregionally advanced nasopharyngeal carcinoma. J Cancer Res Clin Oncol 2013, 139:1063-1071.
• [12]Ma BB, Kam MK, Leung SF, Hui EP, King AD, Chan SL, Mo F, Loong H, Yu BK, Ahuja A, Chan AT: A phase II study of concurrent cetuximab-cisplatin and intensity-modulated radiotherapy in locoregionally advanced nasopharyngeal carcinoma. Ann Oncol 2012, 23:1287-1292.
• [13]Hirose T, Horvitz HR: An Sp1 transcription factor coordinates caspase-dependent and -independent apoptotic pathways. Nature 2013, 500:354-358.
• [14]Karlseder J, Rotheneder H, Wintersberger E: Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F. Mol Cell Biol 1996, 16:1659-1667.
• [15]Black AR, Jensen D, Lin SY, Azizkhan JC: Growth/cell cycle regulation of Sp1 phosphorylation. J Biol Chem 1999, 274:1207-1215.
• [16]Khan S, Abdelrahim M, Samudio I, Safe S: Estrogen receptor/Sp1 complexes are required for induction of cad gene expression by 17beta-estradiol in breast cancer cells. Endocrinology 2003, 144:2325-2335.
• [17]Duttenhoefer F, Biswas SK, Igwe JC, Sauerbier S, Bierhaus A: Sp1-dependent regulation of PPARalpha in bone metabolism. Int J Oral Maxillofac Implants 2014, 29:e107-e116.
• [18]Chen Y, Huang Y, Huang Y, Xia X, Zhang J, Zhou Y, Tan Y, He S, Qiang F, Li A, Re OD, Li G, Zhou J: JWA suppresses tumor angiogenesis via Sp1-activated matrix metalloproteinase-2 and its prognostic significance in human gastric cancer. Carcinogenesis 2014, 35:442-451.
• [19]Wei D, Wang L, He Y, Xiong HQ, Abbruzzese JL, Xie K: Celecoxib inhibits vascular endothelial growth factor expression in and reduces angiogenesis and metastasis of human pancreatic cancer via suppression of Sp1 transcription factor activity. Cancer Res 2004, 64:2030-2038.
• [20]Hsu TI, Wang MC, Chen SY, Yeh YM, Su WC, Chang WC, Hung JJ: Sp1 expression regulates lung tumor progression. Oncogene 2012, 31:3973-3988.
• [21]Jungert K, Buck A, von Wichert G, Adler G, Konig A, Buchholz M, Gress TM, Ellenrieder V: Sp1 is required for transforming growth factor-beta-induced mesenchymal transition and migration in pancreatic cancer cells. Cancer Res 2007, 67:1563-1570.
• [22]Abdelrahim M, Samudio I, Smith R 3rd, Burghardt R, Safe S: Small inhibitory RNA duplexes for Sp1 mRNA block basal and estrogen-induced gene expression and cell cycle progression in MCF-7 breast cancer cells. J Biol Chem 2002, 277:28815-28822.
• [23]Chiefari E, Brunetti A, Arturi F, Bidart JM, Russo D, Schlumberger M, Filetti S: Increased expression of AP2 and Sp1 transcription factors in human thyroid tumors: a role in NIS expression regulation? BMC Cancer 2002, 2:35.
• [24]Jiang NY, Woda BA, Banner BF, Whalen GF, Dresser KA, Lu D: Sp1, a new biomarker that identifies a subset of aggressive pancreatic ductal adenocarcinoma. Cancer Epidemiol Biomarkers Prev 2008, 17:1648-1652.
• [25]Wang L, Wei D, Huang S, Peng Z, Le X, Wu TT, Yao J, Ajani J, Xie K: Transcription factor Sp1 expression is a significant predictor of survival in human gastric cancer. Clin Cancer Res 2003, 9:6371-6380.
• [26]Li L, Davie JR: The role of Sp1 and Sp3 in normal and cancer cell biology. Ann Anat 2010, 192:275-283.
• [27]Majello B, De Luca P, Suske G, Lania L: Differential transcriptional regulation of c-myc promoter through the same DNA binding sites targeted by Sp1-like proteins. Oncogene 1995, 10:1841-1848.
• [28]Marampon F, Casimiro MC, Fu M, Powell MJ, Popov VM, Lindsay J, Zani BM, Ciccarelli C, Watanabe G, Lee RJ, Pestell RG: Nerve growth factor regulation of cyclin D1 in PC12 cells through a p21RAS extracellular signal-regulated kinase pathway requires cooperative interactions between Sp1 and nuclear factor-kappaB. Mol Biol Cell 2008, 19:2566-2578.
• [29]Pore N, Liu S, Shu HK, Li B, Haas-Kogan D, Stokoe D, Milanini-Mongiat J, Pages G, O’Rourke DM, Bernhard E, Maity A: Sp1 is involved in Akt-mediated induction of VEGF expression through an HIF-1-independent mechanism. Mol Biol Cell 2004, 15:4841-4853.
• [30]Chen YJ, Chang WM, Liu YW, Lee CY, Jang YH, Kuo CD, Liao HF: A small-molecule metastasis inhibitor, norcantharidin, downregulates matrix metalloproteinase-9 expression by inhibiting Sp1 transcriptional activity in colorectal cancer cells. Chem Biol Interact 2009, 181:440-446.
• [31]Chuang JY, Wu CH, Lai MD, Chang WC, Hung JJ: Overexpression of Sp1 leads to p53-dependent apoptosis in cancer cells. Int J Cancer 2009, 125:2066-2076.
• [32]Wang HB, Liu GH, Zhang H, Xing S, Hu LJ, Zhao WF, Xie B, Li MZ, Zeng BH, Li Y, Zeng MS: Sp1 and c-Myc regulate transcription of BMI1 in nasopharyngeal carcinoma. FEBS J 2013, 280:2929-2944.
• [33]Zhao WF, Wang HB, Xie B, Hu LJ, Xu LH, Kuang BH, Li MZ, Zhang X: Sp1 and Sp3 are involved in the full transcriptional activity of centromere protein H in human nasopharyngeal carcinoma cells. FEBS J 2012, 279:2714-2726.
• [34]Song LB, Zeng MS, Liao WT, Zhang L, Mo HY, Liu WL, Shao JY, Wu QL, Li MZ, Xia YF, Fu LW, Huang WL, Dimri GP, Band V, Zeng YX: Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma progression and immortalizes primary human nasopharyngeal epithelial cells. Cancer Res 2006, 66:6225-6232.
• [35]Willoughby JA Sr, Sundar SN, Cheung M, Tin AS, Modiano J, Firestone GL: Artemisinin blocks prostate cancer growth and cell cycle progression by disrupting Sp1 interactions with the cyclin-dependent kinase-4 (CDK4) promoter and inhibiting CDK4 gene expression. J Biol Chem 2009, 284:2203-2213.
• [36]Grinstein E, Jundt F, Weinert I, Wernet P, Royer HD: Sp1 as G1 cell cycle phase specific transcription factor in epithelial cells. Oncogene 2002, 21:1485-1492.
• [37]Kavurma MM, Khachigian LM: Sp1 inhibits proliferation and induces apoptosis in vascular smooth muscle cells by repressing p21WAF1/Cip1 transcription and cyclin D1-Cdk4-p21WAF1/Cip1 complex formation. J Biol Chem 2003, 278:32537-32543.
• [38]Kong QL, Hu LJ, Cao JY, Huang YJ, Xu LH, Liang Y, Xiong D, Guan S, Guo BH, Mai HQ, Chen QY, Zhang X, Li MZ, Shao JY, Qian CN, Xia YF, Song LB, Zeng YX, Zeng MS: Epstein-Barr virus-encoded LMP2A induces an epithelial-mesenchymal transition and increases the number of side population stem-like cancer cells in nasopharyngeal carcinoma. PLoS Pathog 2010, 6:e1000940.
• [39]Liao WT, Song LB, Zhang HZ, Zhang X, Zhang L, Liu WL, Feng Y, Guo BH, Mai HQ, Cao SM, Li MZ, Qin HD, Zeng YX, Zeng MS: Centromere protein H is a novel prognostic marker for nasopharyngeal carcinoma progression and overall patient survival. Clin Cancer Res 2007, 13:508-514.
• [40]Guo XZ, Zhang G, Wang JY, Liu WL, Wang F, Dong JQ, Xu LH, Cao JY, Song LB, Zeng MS: Prognostic relevance of centromere protein H expression in esophageal carcinoma. BMC Cancer 2008, 8:233.
• [41]Liao WT, Feng Y, Li ML, Liu GL, Li MZ, Zeng MS, Song LB: Overexpression of centromere protein H is significantly associated with breast cancer progression and overall patient survival. Chin J Cancer 2011, 30:627-637.
• [42]Calao M, Sekyere EO, Cui HJ, Cheung BB, Thomas WD, Keating J, Chen JB, Raif A, Jankowski K, Davies NP, Bekkum MV, Chen B, Tan O, Ellis T, Norris MD, Haber M, Kim ES, Shohet JM, Trahair TN, Liu T, Wainwright BJ, Ding HF, Marshall GM: Direct effects of Bmi1 on p53 protein stability inactivates oncoprotein stress responses in embryonal cancer precursor cells at tumor initiation. Oncogene 2013, 32:3616-3626.
• [43]Fasano CA, Dimos JT, Ivanova NB, Lowry N, Lemischka IR, Temple S: shRNA knockdown of Bmi-1 reveals a critical role for p21-Rb pathway in NSC self-renewal during development. Cell Stem Cell 2007, 1:87-99.
• [44]Bruggeman SW, Hulsman D, Tanger E, Buckle T, Blom M, Zevenhoven J, van Tellingen O, van Lohuizen M: Bmi1 controls tumor development in an Ink4a/Arf-independent manner in a mouse model for glioma. Cancer Cell 2007, 12:328-341.
• [45]Gao Y, Jia Z, Kong X, Li Q, Chang DZ, Wei D, Le X, Suyun H, Huang S, Wang L, Xie K: Combining betulinic acid and mithramycin a effectively suppresses pancreatic cancer by inhibiting proliferation, invasion, and angiogenesis. Cancer Res 2011, 71:5182-5193.
• [46]Kennedy BJ, Torkelson JL: Long-term follow-up of stage III testicular carcinoma treated with mithramycin (plicamycin). Med Pediatr Oncol 1995, 24:327-328.
• [47]Stamp TC, Child JA, Walker PG: Treatment of osteolytic myelomatosis with mithramycin. Lancet 1975, 1:719-722.
• [48]Yuan P, Wang L, Wei D, Zhang J, Jia Z, Li Q, Le X, Wang H, Yao J, Xie K: Therapeutic inhibition of Sp1 expression in growing tumors by mithramycin a correlates directly with potent antiangiogenic effects on human pancreatic cancer. Cancer 2007, 110:2682-2690.